Project 3: Immunopharmacology of FVIII Bioengineering and Gene Therapy Summary The development of inhibitory antibodies directed against human factor VIII remains the most significant clinical complication associated with the treatment of hemophilia A and is a critical barrier to gene therapy approaches. Therefore, improved understanding of the immunology and pharmacology related to promising liver-directed adeno-associated viral (AAV) vector and hematopoietic stem cell (HSC)-directed lentiviral vector (LV) gene therapy strategies is needed. The studies proposed in Project 3 of this U54 application are designed to advance our knowledge of fVIII immunobiology in the context of these gene therapies. The proposed studies are designed to i) analyze the molecular composition and immune reactivity of bioengineered recombinant infusion products as well as liver-directed AAV and HSC-directed LV gene therapy derived fVIII, ii) identify critical design parameters associated with immunogenicity and inhibitor eradiation potential of liver-directed AAV-fVIII gene therapy and iii) evaluate the immunogenicity and inhibitor eradicating potential of non-genotoxic HSC-directed LV gene therapy protocols. These studies will take advantage of available fVIII, glycomics, structural biology, immunology and gene therapy expertise as well as diverse state-of-the-art technologies, including our recently described in silico codon-optimization, synthetic promoter engineering and ancestral sequence reconstruction technology, to improve the current understanding and better assess the overall potential for gene therapy to address all hemophilia A patient populations including those with fVIII inhibitors.